Author summary We manually curated a set of 255 splice events detected in a large-scale tissue-based proteomics experiment and found that more than a third had evidence of significant tissue-specific differences. Events that were significantly tissue-specific at the protein level were highly conserved; almost 75% evolved over 400 million years ago. The tissues in which we found most evidence for tissue-specific splicing were nervous tissues and cardiac tissues. Genes with tissue-specific events in these two tissues had functions related to important cellular structures in brain and heart tissues. These splice events may have been essential for the development of vertebrate heart and muscle. However, our data set may not be representative of alternative exons as a whole. We found that most tissue specific splicing was strongly conserved, but just 5% of annotated alternative exons in the human gene set are ancient. More than three quarters of alternative exons are primate-derived. Although the analysis does not provide a definitive answer to the question of the functional role of alternative splicing, our results do indicate that alternative splice variants may have played a significant part in the evolution of brain and heart tissues in vertebrates. The role of alternative splicing is one of the great unanswered questions in cellular biology. There is strong evidence for alternative splicing at the transcript level, and transcriptomics experiments show that many splice events are tissue specific. It has been suggested that alternative splicing evolved in order to remodel tissue-specific protein-protein networks. Here we investigated the evidence for tissue-specific splicing among splice isoforms detected in a large-scale proteomics analysis. Although the data supporting alternative splicing is limited at the protein level, clear patterns emerged among the small numbers of alternative splice events that we could detect in the proteomics data. More than a third of these splice events were tissue-specific and most were ancient: over 95% of splice events that were tissue-specific in both proteomics and RNAseq analyses evolved prior to the ancestors of lobe-finned fish, at least 400 million years ago. By way of contrast, three in four alternative exons in the human gene set arose in the primate lineage, so our results cannot be extrapolated to the whole genome. Tissue-specific alternative protein forms in the proteomics analysis were particularly abundant in nervous and muscle tissues and their genes had roles related to the cytoskeleton and either the structure of muscle fibres or cell-cell connections. Our results suggest that this conserved tissue-specific alternative splicing may have played a role in the development of the vertebrate brain and heart.